Quantitative analysis of the collagens in the bovine cornea

Calf corneas were treated with pepsin at 4°C in acetic acid and 95% of the tissue was solubilized. The resulting preparation was separated into major collagen fractions by salting out at neutral pH and these fractions were sub-fractionated as necessary to provide a quantitative estimate of the different types of collagen in the tissue. The collagen types were identified by electophoretic analysis of the α-chains and cyanogen bromide peptides. The major component was type I collagen which comprised up to 94% of the solubilized preparation; type II and III collagen were not detected; a fraction which yielded type VI SLS aggregated comprised at least 6%. Gel electrophoretic and solubility studies on the latter fraction showed it is similar to the collagen fraction found by Burgeson et al. (1976) in placental membranes. Examination of the small, pepsin-resistant residue revealed two additional components which may indicate collagen α-chains different from any of the types identified in this study.     

A comparative study of elasmobranch corneal and scleral collagens

Corneas, dissected from young and adult spiny dogfish sharks (Squalus acanthias), were prepared for transmission electron microscopy and immunofluorescence. In the latter case, tissues were fixed in formaldehyde solutions, sectioned with a cryostat, incubated with antibodies specific for collagen types I and II, and examined by indirect immunofluorescence. Collagen α- and β- chains were separated by sodium dodecylsulfate-slab gel electrophoresis and characterized by two-dimensional mapping of ¹²⁵I-labeled peptides generated by tryptic and chymotryptic digestion.The corneal stroma, the sutural fibers which span the stroma, and the surrounding limbus were positive for type I collagen, as judged by immunofluorescence. The corneal stroma was negative for type II collagen. Scleral cartilage matrix was intensely positive for type II collagen, but was negative for type I. In addition, the perichondrium of the scleral cartilage was positive for type I collagen. In confirmation of these results, slab gel electrophoresis revealed α1, and α2-like bands from shark corneal stroma, but only an α1-like band from shark cartilage collagen. Two-dimensional peptide mapping revealed some degree of resemblance between the α1 band of shark corneal stroma and the α1 band of chick type I collagen. Likewise, the α1 band of shark cartilage collagen somewhat resembled the α1 band of chick type II collagen. The α2-like band of shark corneal stroma did not closely resemble the α2 band of chick type I collagen. The most prominent β band of shark corneal stroma appeared to be a dimer composed of one α1 chain and one α2 chain. The collagen of shark corneal stroma was very susceptible to degradation by pepsin, whereas that from shark cartilage was much less susceptible.     

Organization of collagen types I and V in the embryonic chicken cornea

The distribution and organization of type I and type V collagens were studied in the embryonic chicken cornea using anti-collagen, type specific, monoclonal antibodies and immunoelectron microscopy. These studies were performed on lathyritic 17-day corneas treated at 4 degrees C or 37 degrees C. At the lower temperature, collagen fibril structure is disrupted; at the higher temperature, normal fibril structure is maintained. Corneas from non-lathyritic 17-day chick embryos, reacted at the two different temperatures, were studied for comparison. In Bowman's membrane, the thin (20 nm) fibrils were labelled by antibodies against both type I and type V collagen under all conditions studied. In the corneal stroma, the striated collagen fibrils (25 nm) were labelled with the antibodies against type I collagen in all cases, and by antibodies against type V collagen under conditions where fibril structure was disrupted. These results are consistent with the concept of heteropolymeric fibrils consisting of both type I and type V collagen molecules assembled such that the epitopes on the type V molecule are unavailable to antibody unless the fibrillar structure is disrupted. We suggest that the interaction of type V collagen with type I collagen may be responsible for the small diameter fibrils and the rigid control of fibril structure found in the cornea.     

The collagens of the developing bovine cornea

The morphology of the developing bovine eye has been examined and the collagens in fetal bovine eyes from three months' gestation to maturity have been solubilized by pepsin treatment and analyzed to determine the ratios of the predominant types of collagen. The type I collagen decreased, while the type V collagen increased with age. Type III collagen comprised less than 1% of all the corneas, except for the three-month fetal calf. The anterior to posterior thickness of the paraffin-embedded fetal calf cornea increased from the third to the seventh month, decreased from the seventh month to birth, and then increased after birth. Descemet's membrane increased in thickness with age. Analysis of dissected regions of the calf cornea showed a uniform distribution of the collagen populations from the center to the limbus (89% type I, 10% type V and less than 1% type III collagen) and uniformity through the depth of the stroma, except that type III was concentrated around Bowman's layer, and type IV in Descement's membrane. The localization of the different collagens was consistent with the immunofluorescent staining studies with anticollagen antibodies, but the ratios of the intensities of the fluorescence did not correspond to the quantitative analyses. These results are concordant with other studies that have shown that antibody binding may be masked or diminished in certain tissues and therefore immunofluorescence cannot be used reliably for quantitative measurements.     

IGF-II and collagen expression by keratocytes during postnatal development

Keratocytes produce the extensive stromal matrix of the cornea during the late embryonic and neonatal time periods. We propose to test the hypothesis that their biosynthetic activity declines during this process. Keratocytes were isolated from corneas of 6-8-week-old rabbits and corneas of 1-2-year-old cows and their ability to proliferate and synthesize collagen in serum-free media was determined. Rabbit keratocyte cultures increased 38% in DNA content after one week and deposited collagen type I and IGF-II in the media. Bovine keratocyte cultures, in contrast, did not increase in DNA or produce detectable collagen and IGF-II. Bovine keratocytes cultured in media previously conditioned by rabbit keratocytes, however, increased 56% in DNA content, and deposited collagen type I into the media. Microarray analysis of mRNA from neonatal and adult mouse keratocytes was used to confirm these differences. Compared to adult mouse keratocytes, neonatal keratocytes showed high expression levels of IGF-I, IGF-II and collagen types III and V. Since previous studies showed that IGFs stimulate bovine keratocytes to proliferate and to synthesize procollagen type I, we therefore propose that the results of this study suggests that the IGFs may play an important role in regulating early corneal growth in vivo.     

Studies on a distinct fraction of bovine vitreous body collagen

We were able to separate pepsin-solubilized collagen of the bovine vitreous body into three distinct fractions by differential salt precipitation with 0.5 M acetic acid containing 0.7 M, 1.2 M, and 2.0 M NaCl, respectively. The 0.7 M NaCl fraction showed (1) an electrophoretic mobility in polyacrylamide gel identical to that of type II collagen, (2) CNBr-derived peptides very similar to type II collagen CNBr-peptides, and (3) a reaggregation behaviour closely related to type II collagen as observed by electron microscopy. Therefore, the fibres of the bovine vitreous body appear to contain a mixture of a collagen very similar to type II collagen precipitable in the presence of 0.7 M NaCl, at least two different collagen types that can be precipitated at 1.2 M, and 2.0 M NaCl concentrations that have not been identified previously.     

Comparative studies of collagens in normal and keratoconus corneas

In this paper we present strong evidence that the aberrations in keratoconus corneas are not directly related to alterations in collagen composition and distribution. This conclusion is based on comparative studies of collagen types I, III, IV, V and the recently described collagen types VI and VII in keratoconus and normal corneas. The data are derived from biochemical analysis of collagen fractions sequentially extracted with pepsin and sodium-dodecylsulphate, from amino acid analysis of hydrolysates of entire corneal tissues as well as from immunoblotting of the extracted collagens with specific antibodies. These antibodies were also used to examine the distribution of the collagens in immunofluorescence experiments on corneal sections. The yields of the collagen extractions were demonstrated to be age dependent but were not altered in keratoconus samples. Apart from one case associated with osteogenesis imperfecta type I, comparative studies of keratoconus and normal corneas showed no differences in collagen composition of the extracts. This was confirmed by amino acid analysis of tissue-hydrolysates. The distributions of collagen types I, III, IV, V, VI and VII in corneal sections were in general unchanged in keratoconus corneas, the only differences being in scar tissues observed in the Bowman layer of some keratoconus samples.     

Immunogold fine structural localization of extracellular matrix components in aged human cornea II. Collagen types V and VI

Using immunogold immunocytochemical techniques we studied the distribution of collagen types V and VI in corneal tissue from seven enucleated human eyes (age range, 63–78 years). Results obtained by cryoultramicrotomy were marginally more intense than those obtained using London Resin white (LR white) embedding. Type V collagen was present in the striated collagen fibrils in Bowman's layer, in the stroma and in a thin, non-banded anterior zone of Descemet's membrane. Our results suggest that types I, IIl and V collagen co-distribute in striated collagen fibrils. By contrast, type VI collagen was located in fine filaments in the interfibrillar matrix of the stroma, in Bowman's layer and in the anchoring plaques of the sub-epithelial basement-membrane complex. This implies an importance in epithelial adhesion which was previously unsuspected. Keratocyte bodies were electron-dense, amorphous extracellular deposits of matrix-like material, and these were labelled with types III, V and VI collagen antibodies. Long-spacing collagen was observed in the corneal stroma, and this deposit did not contain any of the collagen types studied.Supported by the W.H. Ross Foundation for the Prevention of Blindness Offprint requests to: G.E. Marshall     

Collagen synthesis by rabbit neural retina in vitro and in vivo

Monolayer cultures established from explants of adult rabbit neural retina were used for studies of collagen biosynthesis. Preliminary characterization indicates that the cultures consist predominantly of astrocytic glial cells. Type I comprises 90% of the collagen secreted into the medium by these cultures. AB collagen is also produced. Approximately the same proportion of these macromolecules is secreted by explants as by cells through the fifth passage. Although evidence indicates that the neural retina of some avian species contributes to the vitreous matrix during embryogenesis, no type II-related vitreous collagen is found in the products of cultures of this tissue from the adult rabbit.Since glial cells from the retina have been identified in pathological vitreous membranes, it is of interest to determine if they synthesize collagen within the vitreous and if the type of collagen produced is influenced by the vitreous matrix. Neural retina cells propagated in cell culture were injected into the rabbit vitreous and the newly-synthesized collagen was marked with radiolabeled amino acids and analyzed by CNBr peptide analysis. As in cell culture, the major collagen produced by neural retina within the vitreous is type I. The secretion by these potentially invasive cells of a type of collagen not normally found within the vitreous may contribute to vitreous pathology.     

Intravitreal neovascular tissue of proliferative diabetic retinopathy: an immunohistochemical study.

Intravitreal neovascular tissue in 8 cases of proliferative diabetic retinopathy was investigated using immunohistochemical techniques. All 8 cases yielded positive immunoreactivity for type II collagen (vitreous collagen). The intravitreal neovascular tissue was classified into two groups (A or B), depending upon the distribution of type II collagen. In group A (3 cases), blood vessels were entirely surrounded by vitreous collagen, and in group B (5 cases), the vessels proliferated on one side of a mass of vitreous collagen. Type I and III collagens were distributed diffusely within the extracellular space of the tissue, whereas type IV collagen and fibronectin (FN) formed a basement membrane-like foundation for the newly formed vessels. Glial fibrillary acidic protein (GFAP)-immunoreactive cells were not clearly detected in any of the cases. Neovascular tissue typically proliferated on the posterior vitreous surface (as found in group B), but was also found to penetrate the vitreous gel (as found in group A). As neovascular tissue proliferation proceeded, types I, III and IV collagens and FN were produced. Glial cells (GFAP-positive cells) were not essential for neovascular tissue formation.     

Packages of vitreous collagen (type II) in the human retina: an indication of postnatal collagen turnover?

The purpose of this study was to evaluate the vitreoretinal border in the (pre-)equatorial area in nonpathologic human donor eyes, because the majority of retinal defects induced by posterior vitreous detachment (PVD) are located there. Nine eyes (24-80 years) were fixed and embedded in Technovit 8100. After evaluation by light microscope, areas of interest were selected for immunotransmission electron microscope. Anti-type II collagen antibody was used to stain vitreous fibrils and lamellae; anti-type IV collagen antibody was used to identify the internal limiting lamina (ILL); anti-vimentin and anti-CD-68 antibodies stained retinal Muller cells and macrophages, respectively. Observations included fusing of lamellae with the ILL, an intravitreal course of the ILL, and clear focal interruptions in the ILL. In addition, an obvious finding was the presence of intraretinal packages of type II collagen. Interestingly these collagen packages were closely related to Muller cells and, in several eyes, also to macrophages, cell debris and interruptions in the ILL. In our opinion, the collagen packages can reflect the net result of a process of interactive remodelling, in which both breakdown and synthesis of vitreous and ILL collagens take place. Connections between vitreous and intraretinal collagen networks can make the (pre-)equatorial area more vulnerable to tearing and retinal detachment in the case of liquefaction and PVD.     

Age-related changes on the surface of vitreous collagen fibrils

PURPOSE: To determine whether aging vitreous collagen fibrils undergo ultrastructural changes that might underlie vitreous liquefaction and posterior vitreous detachment. METHODS: Vitreous collagen fibrils from 21 human subjects (age range, 3-89 years) and from bovine eyes were isolated on electron microscopy grids. Cupromeronic blue labeling in the presence of 0.3 M MgCl(2) and immunogold labeling for collagen types II and IX were analyzed by transmission electron microscopy. RESULTS: Aging was associated with marked changes on the surface of human vitreous collagen fibrils, including an exponential loss of type IX collagen along with its chondroitin sulfate side-chains (half-life, 11 years) and a fourfold increase in the exposure of type II collagen. CONCLUSIONS: Despite being a minor component of vitreous collagen fibrils, type IX collagen, probably by virtue of its chondroitin sulfate side-chains, shields type II collagen from exposure on the fibril surface. With aging, this shielding diminishes, resulting in the surface exposure of "sticky" type II collagen and thus predisposing the vitreous collagen fibrils to fusion. These changes could underlie vitreous liquefaction and weakening of vitreoretinal adhesion.     

Cellular redox state predicts in vitro corneal endothelial cell proliferation capacity

Cellular redox state using the non-invasive mitochondrial autofluorescence technique of redox fluorometry was evaluated as a predictor for corneal endothelial proliferative capacity in vitro. Human corneal endothelial cells (HCEC) harvested from eye bank corneas were cultured in plates with two different coating substrates; type I collagen and poly-D-lysine. Cellular autofluorescence was measured with both DAPI (excitation: G365, emission: bandpass 445/50) and FITC (excitation: bandpass 450-490, emission bandpass 515-565) filter sets on days 3, 5, 7, and 14. The redox fluorometric ratio was calculated as net "DAPI" signal intensity divided by net "FITC" signal intensity. Normalized redox ratio was calculated as redox ratio divided by individual cell size. Cellular proliferation was analyzed by live cell count on days 2, 7, and 14. Mitochondrial staining was performed on days 4 and 14. The poly-d-lysine substrate decreased the proliferation capacity of HCEC in comparison to type I collagen out to 2 weeks (p=0.045). The cellular redox fluorometric ratio decreased significantly as the cells proliferated (p<0.001). The cells cultured on type I collagen coated plates exhibited significantly lower redox fluorometric ratios than cells cultured on poly-D-lysine coated plates at day 7 (p=0.015). Normalized redox ratio showed significantly lower value in type I collagen coated plates at days 7 (p=0.015) and 14 (p=0.039). Correlated cell proliferation capacity was significantly higher on type I collagen coating at days 7 and 14 (p=0.045 and p=0.049 respectively). HCECs showed different growth potential in vitro on different culture surface coating agents. This difference was well correlated with cellular redox ratios determined using redox fluorometry. Cellular redox ratio can be a potential predictor of cellular proliferation capacity.     

Characterization of collagen from normal human sclera

Human scleral tissue contains approximately 50% collagen by weight, consisting predominantly of type I collagen. There is little or no evidence for the presence of substantial quantities of type II, type III or other collagen types. There appears to be no difference in either collagen content or genetic type in sclera between adult and juvenile tissues or between anterior and posterior segments of the sclera, although, with increased age there is a marked increase both in the extent of glycosylation of the collagen and its resistance to solubilization by treatment with pepsin.     

正常角膜与圆锥角膜中的Ⅰ型、Ⅵ型胶原与金属蛋白酶-2

目的对比正常角膜与圆锥角膜中的Ⅰ型,Ⅵ型胶原及金属蛋白酶-2的定位与含量。方法应用酶免疫组化及间接免疫荧光技术检测正常与圆锥角膜中Ⅰ型、Ⅵ型胶原及金属蛋白酶-2（MMP-2)的定位。阳性结果应用图像分析系统进行定量分析。结果免疫组化结果显示所有角膜中Ⅰ型胶原存在于前弹力层和基质中;Ⅵ型胶原存在于上皮基底膜、前弹力层、基质、后弹力层;MMP-2存在于上皮、基质、内皮中。同正常角膜比,圆锥角膜中Ⅰ型、Ⅵ型胶原的含量无差别而MMP-2的含量明显增多,差异有显著性。结论圆锥角膜的MMP-2含量增多,其所导致的胶原降解异常可能是圆锥角膜发病的关键。     

Histogenesis of the intravitreal membrane and secondary vitreous in the mouse

PURPOSE: The intravitreal membrane (IVM) is a membranous structure between the primary and secondary vitreous bodies in developing mammalian eyes. In this study, for the first time the histogenesis of the IVM and the relationship between the hyaloid vasculature and the IVM was characterized in newborn mice. METHODS: Eyes of mice less than 12 days old were fixed and embedded. From these, serial paraffin-embedded sections were made for lectin histochemistry, immunohistochemistry, and picrosirius red (PSR) staining, and ultrathin sections were made for transmission electron microscopy (TEM). Eight biotinylated lectins and antibodies for laminin and type IV collagen were used. RESULTS: Among the eight lectins tested, concanavalin A (Con A) agglutinin, Ricinus communis agglutinin I, and wheat germ agglutinin demonstrated strong positive staining in the IVM and vitreous fibrils of the primary and secondary vitreous bodies. They also bound to the internal limiting membrane (ILM) of the retina. At postgestational day 4, the secondary vitreous first appeared between the ILM and the vasa hyaloidea propria (VHP). Immunohistochemical staining revealed that the IVM consists of extracellular matrix components including laminin and type IV collagen, whereas PSR staining and TEM showed that collagen fibrils in the IVM are bundled and continuous with the basement membrane of hyaloid capillaries or the VHP. CONCLUSIONS: Lectin histochemistry and immunohistochemistry provided good methods for visualizing the structures of the IVM and vitreous fibrils. These results suggest that the IVM is separated from the basement membrane of the retinal ILM along with the vascular network of the VHP when the secondary vitreous begins to form.     

Glycosaminoglycan and collagen distribution in the developing human vitreous

· Background: We determined the distribution of glycosaminoglycans and collagens in the developing human vitreous. · Methods: Eighty human eyes from 5 gestational weeks to 2 postnatal years of age were used. Glycosaminoglycan components were determined by enzyme digestion with hyaluronidase or chondroitinase AC and ABC and immunohistochemistry for chondroitin, chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate. Collagen distribution was determined by immunohistochemistry for types I, II, and III collagens. · Results: Enzyme digestion showed that throughout development hyaluronic acid is the main glycosaminoglycan in the vitreous and in the extraocular space at 5–7 gestational weeks. Both areas were filled with mesenchymal cells. Immunohistochemistry showed chondroitin-6-sulfate in the vitreous between 6 and 40 gestational weeks, and chondroitin-4-sulfate between 12 and 40 gestational weeks. Hyaluronic acid and chondroitin sulfate appeared in the retina and around the hyaloid vessels at 12–40 weeks. Immunohistochemistry showed type III collagen in the vitreous and around the mesenchymal cells at 5–7 weeks that was replaced by type II collagen after 8 weeks. · Conclusions: Hyaluronic acid is the major glycosaminoglycan in the vitreous throughout development, except for the transient appearance of chondroitin sulfate at 6–40 gestational weeks. Type III is the main collagen in the early developing vitreous that converts to type II collagen at 8 weeks. The primary and secondary vitreous has the same components as these macromolecules. These vitreous glycosaminoglycans and collagens seem to be produced by mesenchymal cells at an early stage and by the retina and hyaloid vessels during middle and late development. Received: 7 November 1997 Revised version received: 5 January 1998 Accepted: 7 January 1998     

Characterization of the Descemet's membrane/posterior collagenous layer isolated from Fuchs' endothelial dystrophy corneas

The combined Descemet's membrane (DM) and posterior collagenous layer (PCL) of Fuchs' endothelial dystrophy corneas were isolated and characterized by biochemical and immunofluorescence methods. The amino acid composition of the Fuchs' DM-PCL was similar to age-matched normal Descemet's membranes (DM). As determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ¹²⁵I two-dimensional peptide mapping, normal DM and Fuchs' DM-PCL contained the same collagen types [type IV and endothelial cell (EC) collagen], but a slight discrepancy was seen in the electrophoretic mobility of some collagen chains. Immunofluorescence staining localized fibrinogen/fibrin to Fuchs' DM-PCL but not to normal DM. These data suggest (1) that the appearance of 110 nm banded material in sheets and fusiform bundles characteristic of Fuchs' PCL is not due to the presence of a new (abnormal) collagen type but may represent altered assembly of collagen molecules, and (2) that the fibrinolytic system may play a role in the degenerative process of Fuchs' endothelial dystrophy.     

Vascular remnants in the rabbit vitreous body. II. Enzyme digestion and immunohistochemical studies

The purpose of this study was to evaluate the composition of ghost vessels and the newly identified intravitreal structures type 1 and 2 (IVS-1 and 2) observed in the rabbit vitreous body. Rabbit eyes (n = 10, 0.5- approximately 36 months of age) were fixed and embedded in plastic. Post-embedding immuno transmission electron microscopy and enzyme digestion methods specifically directed at vascular extracellular matrix components (collagen IV, elastin and hyaluronan) were used in order to confirm the postulated vascular origin of IVS-1 and 2. In addition, markers of vitreous extracellular matrix components (collagen II, hyaluronan) were used. The postulated vascular nature of ghost vessels and IVS-1 was confirmed by a positive labelling with anti-collagen IV, whereas the demonstration of elastin (by anti-elastin antibodies and elastase digestion) in IVS-1 and 2 confirms their arterial origin. These vascular remnants were also labelled with a hyaluronan marker and with anti-collagen II. The presence of remnants of the hyaloid artery system throughout the vitreous matrix is in conflict with a strict spatial separation between the primary and secondary vitreous during embryonic development as proposed in the literature. It strongly supports an alternative theory which suggests an interactive remodelling of this matrix. The presence of hyaluronan in remnants of the hyaloid system is inconclusive, since hyaluronan is a component both of the adult vitreous matrix and of the vascular extracellular matrix. The presence of collagen II in vascular structures is highly interesting, since it supports another challenging theory, which suggests that lamellae develop alongside tracts formerly occupied by the larger hyaloid vessels.     

Quantitative analysis of immunogold labellings of collagen types I,III, IV and VI in healthy and pathological human corneas

Background: We studied the distribution of collagen types I, III, IV and VI in one healthy human cornea and in seven pathological human corneas, in which the disorders were three cases of pseudophakic bullous keratopathy (two severe, one moderate) and one case each of stage IV keratoconus, chronic ulcer, vascularized cornea and disciform keratitis. Methods: Transmission electron microscopy examinations were performed on post-embedding immunogold-labelled sections. The staining was evaluated by gold particle count in the different tissues. The presence or absence of a given antigen was determined by statistical analysis, using a d-value test. Results: Our results on healthy corneal tissues corroborate the data available from previous studies, except for collagen type VI, which we found to be absent in Bowman's layer. In pathological corneas with a collagenous layer posterior to Descemet's membrane, collagen types I, III and especially IV were detected in this collagenous layer. Collagen types I, III and VI were detected in the anterior healed stroma of other pathological corneas, except for the keratoconus cornea, in which intense collagen III staining was observed. Conclusion: The presence of collagen types I and III in the posterior collagenous layer of our pseudophakic bullous keratopathy corneas suggests that this layer corresponds to scar tissue secreted by stimulated endothelial cells.